Affinity Labeling of RNA-Polymerase II in the Transcriptionally Active Complex by a Phosphorylating Analog of the Initiation Substrate

L. K. Savinkova^1*, A. A. Sokolenko¹, V. A. Rau¹, T. V. Arshinova¹, Yu. V. Gerasimova², N. V. Kudryashova², and T. S. Godovikova²

¹Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090 Russia; E-mail: savsok@niboch.nsc.ru

²Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090 Russia

^* To whom correspondence should be addressed.

Received December 14, 1999; Revision received May 30, 2000

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Affinity modification of RNA-polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5´-terminal phosphate group with a 4-N,N-dimethylaminopyridine residue (DMAP-pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super-selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA-polymerase II from modification. The third subunit may be involved in the formation of the substrate-binding site of the enzyme.

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KEY WORDS: RNA-polymerase II, promotor, basal transcription factors, transcription initiation, DMAP-derivative of initiation substrate

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